This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have developed a method to map protein solvent acessible surfaces using hydrogen peroxide and UV light to generate hydroxyl radicals. Although the early efforts made use of continuous light source, we recently moved beyond that and developed an ultrafast reaction method to probe protein solvent-accessible surfaces by using a pulsed laser and a quencher. This approach is capable of following reaction times on the order of 100 nanoseconds and slower--faster than any protein known unfolding event. Specifically, the method circumvents problems that reaction with OH (protein oxidation) could cause protein unfolding and oxidation of sites that are not accessible in the native protein, giving misleading results. We avoid unwanted oxidation by using a 248-nm KrF excimer laser to cleave hydrogen peroxide at low concentrations (15 mM, 0.04%), affording hydroxyl radicals that modify the protein in less than a microsecond. In the presence of a scavenger, the radical lifetimes decrease to 1 microsecond, yet the reaction timescales are sufficient to provide significant oxidation of the protein. These times are arguably faster than super-secondary protein structure can unfold as a result of the modification. The radical formation step takes place in a nanoliter flow cell so that only one laser pulse irradiates each bolus of sample. The oxidation sites are located using standard analytical proteomics, requiring less than a nanomole of protein.